Abstract：To enable industrial Saccharomyces cerevisiae ferment xylose into ethanol by the expression of XylA and overexpression of XKS1 and PPP genes TAL1、TKL1、RKI1 and RPE1, the promoter FBA cloned from S. cerevisiae was ligated with xylose isomerase gene (XylA) and the ligation product was inserted into the shuttle expression vector of pYES2, then the expression vector pYES2-FBA-XylA was constructed, and the promoter TPI cloned from S. cerevisiae was inserted into the vectors pUG6 and pUG72, respectively, and then the vector pUG6-TPI and pUG72-TPI were constructed. The vector pUG6-TPI contained the selection marker of KanMX and pUG72-TPI contained the selection marker of ura. Different primer which contained homologous sequences were designed. Taking pUG6 and pUG72 as template, the fragments which enhanced expression of XKS1 and PPP genes TAL1、TKL1、RKI1 and RPE1 were obtained by PCR. The fragment was integrated into S. cerevisiae by the LiAc chemical method. Then the fragment with different selection marker was integrated into the allele loci of S. Cerevisiae. These two different markers were removed by inducing recombination enzyme expression after vector pSH65 were transformed into S. Cerevisiae. The TPI promoter were inserted into the rest 4 loci of the genes and removed the markers in the same way. Then the PPP enhanced recombinant strain was obtained. The expression vector pYES2-FBA-xylA was transformed into the PPP enhanced recombinant, and the recombinant of pXI-T1308U- was constructed. Expression levels of XKS1 and PPP genes between the original strain and recombinant strain were analyzed by qRT-PCR, and the results showed that the transcription levels of TAL1、TKL1、 RKI1、RPE1 and XKS1 of recombinant strain were increased to 1.62, 3.98, 17.36, 4.17 and 4.08-fold, respectively, compared with original strain. XI (EC 5.3.1.5) activity in the cell extracts of the strains were determined, and the recombinant strain exhibited specific XI activity as high as 0.39 U/mg protein whereas no XI activity could be detected in the original strain. The co-fermentation of glucose and xylose of recombinant strain was studied, and fermentation samples were analyzed by HPLC, results showed an increased consumption rate of xylose about 17.54% compared with its original strain. And the ethanol yield was about 0.23 g/g xylose. In conclusion, the recombinant industrial strain could ferment xylose by enhancing expression of the key gene XKS1 and PPP gene under the expression of XI. This work provides a new promising reference for further development of xylose-fermenting yeast for industrial lignocellulosic ethanol production.
